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United States Patent |
5,621,072
|
Watanabe
,   et al.
|
April 15, 1997
|
Purified guaiacum resin and method for making same
Abstract
A purified guaiacum resin which is obtained by isolation from a natural
guaiacum resin with chromatography using a gel for reversed chromatography
as a stationary phase and a polar solvent as a mobile phase. The purified
guaiacum resin does not contain substances causing the repellency of a
specimen or constituents exhibiting unstable color development when it is
used as a body fluid inspection agent and is subjected to color reaction
in the presence of a peroxidase and hydrogen peroxide.
Inventors:
|
Watanabe; Masanao (Tokyo-To, JP);
Kuroda; Kohji (Chofu, JP);
Fujita; Yoshiko (Tokyo-To, JP)
|
Assignee:
|
Dai Nippon Insatsu Kabushiki Kaisha (Tokyo, JP)
|
Appl. No.:
|
418857 |
Filed:
|
April 7, 1995 |
Foreign Application Priority Data
| Aug 17, 1988[JP] | 63-204525 |
Current U.S. Class: |
530/200; 422/55; 422/56; 422/57; 422/61 |
Intern'l Class: |
C11D 015/00; G01N 021/00 |
Field of Search: |
530/200
422/56,55,57,61
|
References Cited
U.S. Patent Documents
2981606 | Apr., 1961 | Keston | 435/14.
|
4297271 | Oct., 1981 | Guthlein et al. | 435/14.
|
Foreign Patent Documents |
63-223095 | Sep., 1988 | JP.
| |
63-223096 | Sep., 1988 | JP.
| |
8707718 | Dec., 1987 | WO.
| |
Other References
"Arch. Pharmz." 299, pp. 618-626 (1966) with English AB.
"Arch. Pharmz." 302, pp. 545-554 (1969) with English AB.
|
Primary Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Birch, Stewart, Kolasch & Birch, LLP
Parent Case Text
This application is a continuation of application Sr. No. 08/138,181 filed
on Oct. 20, 1993, now abandoned, which is a continuation of application
Ser. No. 07/803,551 filed Dec. 9, 1991 and now abandoned, which is a
divisional of application Ser. No. 07/242,690, filed Sep. 9, 1988, which
issued as U.S. Pat. No. 5,093,082 on Mar. 3, 1993.
Claims
What is claimed is:
1. A method for preparing a purified guaiacum resin comprising:
subjecting guaiacum resin to reversed phase chromatography using a fixed
gel phase for development with an initial developing solvent of higher
polarity than a solvent of water and methanol at a mixing ratio by volume
of 1:9 so as to eliminate an initially eluted non-color-developing
constituent group and a color-developing unstable constituent group from
the guaiacum resin;
collecting a solution containing a hydrophilic color-developing constituent
group by the use of a solvent having polarity lower than the initial
developing solvent; and
removing the solvent from said collected solution.
2. The method according to claim 1, wherein the elution of the hydrophilic
color-developing constituent is effected by the use of methanol.
3. The method according to claim 1, wherein the elution of the hydrophilic
color-developing constituent is effected by the use of ethanol.
4. The method according to claim 1, wherein the elution of the hydrophilic
color-developing constituent is effected by the use of a solvent of water
and acetonitrile at a mixing ratio of 4:6 by volume.
5. A method for the preparation of a hydrophilic color-developing guaiacum
resin constituent comprising the following steps:
subjecting a guaiacum resin to reverse phase chromatography on a fixed gel
phase by development with a first developing solvent of higher polarity
than a solvent of water and methanol in a mixing ratio of 1:9 (by volume)
to eliminate a non-color-developing constituent and a color developing
unstable constituent;
developing with a second developing solvent having a polarity lower than
the first developing solvent to collect a second elution containing a
hydrophilic color-developing resin constituent group; and
isolating the color-developing resin constituent group from the second
developing solvent.
Description
FIELD OF THE INVENTION
This invention relates to a purified guaiacum resin which is suitable for
use as an oxidation-susceptible color indicator of a diagnostic agent for
inspection of glucose and occult blood in body fluids and also to a method
for preparing the same.
RELATED ART STATEMENT
For detection in early stages, diagnosis and control of diabetes, it is
essential to rapidly and simply determine an amount of glucose in a body
fluid such as urine, blood or lymph.
In prior detection of glucose in body fluids and particularly, urine, it is
the common practice to use an inspection piece in which a glucose
inspection reagent is impregnated in filter paper for judgement with a
degree of color development of an oxidation-susceptible color indicator,
or an inspection piece in which the reagent composition is applied onto a
substrate. These inspection pieces or articles are advantageous from the
standpoint of application in that the inspection operation is simple and
the judgement can be made within a short time. In U.S. Pat. No. 2,981,606,
there is described use of o-tolidine, guaiacum resin and
o-phenylenediamine as an oxidation-susceptible color indicator of
diagnostic agents which are used to detect glucose and occult blood in
body fluids. Since benzidine compounds exhibit high detection sensitivity,
o-tolidine has been predominantly used up to now. Although o-tolidine has
high sensitivity, it is liable to discolor or lower in sensitivity during
storage and a blue substance formed by the color reaction is unstable, so
that a time for accurate reading becomes short, disenabling accurate
judgement in practical applications. Thus, there is a demand for
overcoming the drawback. The guaiac or guaiacum resin ordinarily employed
as another oxidation-susceptible color indicator is a natural product and
is collected from the heartwood of guaiac wood which is a tropical plant.
Accordingly, the guaiacum contains a plurality of impurities other than
constituents effective for coloration or color development, e.g.
alpha-guaiaconic acid, beta-guaiaconic acid, guaiaretic acid, guaiacic
acid, resene, gums, and essential oils and the like as is known in the
art. In addition, the compositions of the effective constituents and the
impurities are not always constant, thus making it difficult to ensure
stable properties such as sensitivity and uniformity in color reaction.
Several methods for purifying the guaiacum resin have been proposed. For
instance, there is known a method in which when the guaiac is subjected to
successive solvent extractions with petroleum ether, an ether and an
alcohol, there is present in the ether-extracted fraction a constituent
capable of developing a blue color in the presence of a peroxidase and
hydrogen peroxide; and three main constituents are analytically obtained
by isolation of the fraction with thin layer chromatography [(H. Auterhoff
et al, "Arch. Pharmz." 299, pp. 618-626 (1966) and 302, pp. 545-554
(1969)]. In U.S. Pat. No. 4,297,271, it is stated that one of the above
constituents has an Rf value of 0.45 in the thin layer chromatography
(toluene/dioxane/glacial acetic acid 90:25:10) and can be industrially
obtained by column chromatography using neutral silica gel and a mixture
of n-heptane/acetic ester. This is described for elucidating a prior art
technique. As described in the patent publication, the guaiac constituent
obtained by the technique is not a single substance but is obtained as a
mixing composition containing non-color-developing substances. From this,
it is believed that because of the instability of the guaiacum resin
constituent composition, the patent indicates the applicability of the
composition at a level of examination of occult blood in the feces where a
simple decision is made to qualitatively determine the presence or absence
of occult blood. For using the guaiacum constituents as an inspection
agent of high performance for glucose and occult blood in body fluids
which requires semi-quantitative determination, the constituents should
have a high sensitivity sufficient for color development at low
concentrations and the capability of varying a developed color density in
a stepwise manner over a range of from low to high concentrations. As is
different from the case of the occult blood in the feces, with liquid
specimens such as body fluids, a reagent layer containing an inspection
agent has to be uniformly wetted with the specimen. In the method
described in the U.S. Pat. No. 4,297,271 it is not possible to remove
substances impeding the wetting properties from the guaiacum resin, with
attendant vital drawbacks of color shading and the instability of an
ultimate color density caused by the non-uniform deposition on or
infiltration into the reagent layer of the specimen.
Moreover, the guaiacum resin contains constituent groups capable of
developing a color but having Rf values of the thin layer chromatography
different from the Rf value of 0.45. Among these groups, there are
included constituents having different regions of color density. In order
to cause quantitatively different color densities to develop over a wide
range of concentration, it is necessary to contain these constituents. In
this connection, however, according to the method described in the above
patent, impurities are contained in large amounts since constituents
covering a wide Rf value of from 0.2 to 0.6 are contained. Thus,
purification is not substantially carried out. At least in an industrial
sense, there is not known any method of recovering a constituent group
effective as an oxidation-susceptible color indicator.
OBJECT AND SUMMARY OF THE INVENTION
The present invention contemplates to solve the above problems involved in
the known color indicators and has for its object the provision of a
purified guaiacum resin which is suitable as an oxidation-susceptible
color indicator of a diagnostic agent for detecting glucose and occult
blood in body fluids because color development-inhibiting substances have
been removed therefrom and it contains substantially effective
constituents, The invention also provides a method for preparing the
purified guaiacum resin.
The present invention relates to a purified guaiacum resin which is
obtained by isolation from a natural guaiacum resin with chromatography
using a gel for reversed chromatography as a stationary phase and a polar
solvent as a mobile phase, The purified guaiacum resin substantially does
not contain color development-inhibiting substances causing the repellency
of a specimen or constituents exhibiting unstable color development when
it is impregnated in filter paper or applied to a substrate for use as a
body fluid inspection agent and is subjected to color reaction in the
presence of a peroxidase and hydrogen peroxide, In addition, the purified
guaiacum resin contains a plurality of color-developing constituents
capable of developing a color over a wide concentration range.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view showing an elution pattern of a crude guaiacum resin
separated by column chromatography; and
FIG. 2 is a view showing an elution pattern of a crude guaiacum resin
obtained by the use of an automatic collection-type chromatograph.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The purified guaiacum resin according to the invention is obtained by
subjecting a natural guaiacum resin to reversed chromatography using a
fixed gel bed and an initial developing solvent of polarity higher than a
solvent of water and methanol at a mixing ratio by volume of 1:9 to
develop and eliminate a non-color developing constituent group including
water-repellent constituents and an unstable color-developing constituent
group from the guaiacum resin, collecting the resultant solution of a
hydrophilic color-developing constituent group, and removing the solvent
from the solution.
When the natural guaiacum resin is isolated according to the above method,
four constituent groups are isolated. The first constituent group contains
small amounts of color-developing constituents which are so small as not
to be used in practical applications and also contains water-repellent
constituents. Although it may be apparently curious that the first
constituent group contains water-repellent substances, this is considered
as follows: for the fabrication of an inspection article, the substances
adsorb on polar carrier of the inspection articles so that the hydrophobic
groups are oriented as arranged to be outward.
The second constituent group comprises reddish purple color-developing
constituents and are unstable, and should be removed.
The third constituent group comprises a plurality of effective constituents
capable of developing a deep blue color and is hydrophilic in nature.
The fourth constituent group comprises a mixture of constituents capable of
developing a color in a wide range of concentration and contain
color-developing constituents with high sensitivity although not
relatively high in concentration. This group is hydrophilic.
The purified guaiacum resin of the invention consists of the third and
fourth constituent groups among the above four groups. The present
invention is described in more detail. In the preparation of the purified
guaiacum resin, a natural guaiacum resin is first dissolved in acetone, to
which toluene is added in order to remove a formed precipitate. The
resultant filtrate is evaporated to dryness and the residue is dissolved
in a polar solvent to obtain a crudely purified guaiacum resin. Examples
of the gel for reversed chromatograph include an alkylated silica gel
obtained by reaction of silanol groups on the surface of divided or
spherical silica gel with an alkyl or aryl chlorosilane, a chemically
combined silica gel, polymer-based alkylated hard gels, and the like. More
particularly, there are mentioned alkylated silica gels such as of
C.sub.18, C.sub.8, C.sub.4, C.sub.2, C.sub.1 and the like, chemically
combined silica gels with terminal groups such as --NH.sub.2, --CN, phenyl
and the like, and polymer-based alkylated hard gels such as of polymeric
C.sub.18 compounds. Although the elution time, more or less, varies
depending upon the type of gel, little difference in the order of elution
of the constituent groups is experienced, ensuring effective isolation of
the groups.
The polar solvents useful in the present invention are selected from
appropriate combinations of ethanol, methanol, ethanolamine, ethylene
glycol, formamide, water, acetonitrile, dimethylsulfoxide,
1,3-dicyanopropane, diethyl ether, propyl chloride, ethyl acetate,
propylamine, ethyl bromide, CHCl.sub.3, tetrahydrofuran, methyl acetate,
acetone, CH.sub.2 Cl.sub.2, 1,2-dichloroethane, sodium octanesulfonate,
dioxane, pyridine, benzonitrile, nitromethane, nitrobenzene and the like.
In some cases, up to 50% of non-polar solvents may be added to the polar
solvent. For controlling the pH, acetic acid, trifluoroacetic acid, sodium
octanesulfonate and the like may be used, but since they should be
separated after elution, there is little chance of adding such materials
except the case where they may be used as coexisting.
In the practice of the invention, the stationary and mobile phases as
described before are used to separate by elution of the first and second
constituent groups, which are color-inhibiting constituents, with an
initial developing solvent. Subsequently, hydrophilic color-developing
constituent groups useful for coloration are eluted. The initial
developing solvent used for the separation of the inhibiting constituents
is preferably one which has a polarity slightly higher than methanol.
Examples of such solvent include mixed solvents of methanol and water,
sodium octanesulfonate, acetic acid, trifluoroacetic acid and the like.
All the mixed solvent should preferably have a polarity higher than that
of a solution of methanol and water at a mixing ratio by volume of 9:1.
The developing solvent used to collect the third and fourth constituent
groups which are color-developing constituent groups is a solvent similar
to the initial developing solvent. The third constituent group may be
collected by means of the initial developing solvent as a constituent of a
longer retention time. In practice, the third and fourth constituent
groups can be eluted by the use of a solvent having a polarity smaller
than the initial developing solvent. The elution with methanol alone or
ethanol is effective.
The purified guaiacum resin obtained according to the invention is suitable
as a color indicator of a test strip for the purpose of detecting glucose
or occult blood in body fluids. It has been found that the guaiacum resin
used for this purpose may contain not only the third and fourth
constituent groups, but also constituents of a longer retention time than
a fraction being eluted without involving any hindrance for achieving the
above purpose.
The body fluid inspection article or piece may be made according to any
known methods. For instance, an ordinary glucose detection composition
obtained by formulating a color indicator, glucose oxidase, a peroxidase,
a pH buffering agent, a water-soluble binder such as polyvinylpyrrolidone,
and, if necessary, a humectant, a sensitivity regulator and a stabilizer
is dissolved in water or a water-alcohol mixture, followed by impregnating
the solution in a filter paper and drying the obtain a test strip. When
the purified guaiacum resin of the invention is applied to the test strip,
the uniformity of color development is improved.
Alternatively, a glucose detection dye containing ink composition in a
non-aqueous solvent which has been proposed recently, is prepared by
dissolving or dispersing in a non-aqueous solvent a color indicator,
glucose oxidase, a peroxidase, a pH buffering agent, a binder and a
water-absorptive powder. After addition of a humectant, a sensitivity
regulator and a stabilizer to the composition, if necessary, the
composition is applied onto a substrate and dried to obtain an inspection
article for glucose detection. This article is better in sensitivity and
stability than the test strip of the type obtained by impregnation into
filter paper. However, when the first constituent group is incorporated
into the guaiacum resin used as the color indicator, water repellency is
liable to appear, with the possibility of non-uniformity. This is
considered as follows: when the non-aqueous solvent is evaporated for
drying in the course of the fabrication of the inspection articles, the
first constituent group locally gathers in the surface layer to impart
water repellency. When the purified guaiacum resin of the invention
consisting of the third and fourth constituent groups is used, the
characteristic features of the guaiacum resin such as high sensitivity and
good color stability are emphasized. The guaiacum resin is especially
effective as the a non-aqueous solvent-based body fluid inspection
article.
In the glucose inspection paper, the glucose in a body fluid reacts with
oxygen in air by the action of a glucose oxidation enzyme such as glucose
oxidase and is finally oxidized into gluconic acid and hydrogen peroxide.
The resultant hydrogen peroxide produces nascent oxygen by the action of
the peroxidase. This oxygen immediately reacts with the guaiacum resin to
cause color development of the indicator. The presence or absence and the
amount of the glucose in the body fluid can be semi-quantitatively
determined in view of the degree of the color development. Accordingly, it
is necessary that the body fluid allow the detection article to be
uniformly wetted so that a density of the color can be judged over a wide
range of from low to high concentrations and that the color tone be stably
held after the color development. In this respect, the purified guaiacum
resin of the invention exhibit good sensitivity, a wide color density
range and good color stability. The purified guaiacum resin of the
invention is effective not only for the above purpose, but also for
detection of hydrogen peroxide and substances capable of producing active
oxygen. The guaiacum resin can be widely employed in the fields of
inspection agents such as in detection of reactions or semi-quantitative
determination in the coexistence of glucose oxidase for glucose, urease
for urea nitrogen, cholesterol oxidase for cholesterol, hemoglobin having
the pseudo-peroxidase activity for occult blood, and the like.
The present invention is described by way of examples, which should not be
construed as limiting the invention.
EXAMPLE 1
Isolation of Purified Guaiacum Resin (1)
100 g of natural guaiacum resin was dissolved in 150 ml of acetone, to
which 1.5 liters of toluene was added under agitation to remove the
resultant precipitate (about 20 g) by filtration by suction. The resultant
filtrate was concentrated in vacuum and dried to obtain about 70 g of a
dried matter in this pretreatment. Part of the dried matter was dissolved
in a mixed solution of methanol/water (65:35) to give a solution of 0.5
g/liter. This solution was once poured 20 .mu.l and then the mixed
solution of methanol/H.sub.2 O (65:35) was passed at a rate of 0.5 ml/min
at 40.degree. C. into a column having a diameter of 4.6 mm and a height of
15 cm and containing octadecylsilane-treated silica gel (C.sub.18) which
had been preliminarily equilibrated with a mixed solution of
methanol/H.sub.2 O (65:35). After confirmation of the elution of first and
second constituent groups, a fraction consisting of a third constituent
group was collected after a retention time of 16 minutes. After 32
minutes, the elution was facilitated by changing the moving bed to
methanol to elute a fourth constituent group up to 60 minutes for
collection. A detector made use of a UV ray with 280 nm.
FIG. 1 shows an elution pattern of a crude guaiacum resin separated by the
column chromatography under the above conditions. The ordinate axis
indicates an absorption of the UV ray at 280 nm and the abscissa axis
indicates a retention time. In the FIG. 1, the first, second, third and
fourth constituent groups are depicted as separated from one another.
After the elution of the third constituent group, the mobile phase was
changed to methanol and the resultant eluate was the fourth constituent
group.
Preparation of Purified Guaiacum Resin (1)
In the same as in the Isolation (1), natural guaiacum resin was pretreated
and 2 liters of a mixed solution of methanol/H.sub.2 O (6:4) was added to
about 70 g of the resultant dried matter.
80 ml of the solution was poured at room temperature into a column having a
diameter of 5 cm and a height of 50 cm and containing
octadecylsilane-treated silica gel (C.sub.18) which had been equilibrated
with a mixed solution of methanol/H.sub.2 O (6:4), followed by passing 100
ml/minute of a mixed solution of methanol/H.sub.2 O (6:4). After 180
minutes at which first and second constituent groups had been eluted,
methanol was flowed to elute and collect a fraction until no absorption of
a UV ray at 280 nm from a detector was observed. The elution pattern is
shown in FIG. 2. The resultant purified guaiacum resin was developed under
the same conditions as in the Isolation (1), whereupon it was confirmed
that the first and second constituent groups were not detected but the
resin consisted of the third and fourth ingredient groups. The amount of
the collected resin was about 0.6 g.
Preparation of Purified Guaiacum Resin (2)
Natural guaiacum resin was pretreated in the same manner as in the
preparation of Purified Guaiacum Resin (1). 2 liters of a mixed solution
of methanol/H.sub.2 O (11:9) was added to about 70 g of the resultant
dried matter. This solution was preliminarily set in a high speed liquid
chromatograph for collection and 100 ml of the solution was once poured at
room temperature into a column having a diameter of 5 cm and a height of
50 cm and containing butyl-chemically combined silica gel (C.sub.4) which
had been equilibrated with a mixed solution of methanol/H.sub.2 O (11:9),
followed by passing 100 ml/minute of a mixed solution of methanol/H.sub.2
O (11:9) to separate by elution of impurities. Thereafter, constituents
contained in the column were eluted with ethanol. For detection, a UV ray
with a wavelength of 280 nm was used. The fraction eluted with the ethanol
was evaporated to dryness in an evaporator to obtain about 0.5 g of
purified guaiacum resin (2).
Preparation of Purified Guaiacum Resin (3)
Natural guaiacum resin was pretreated in the same manner as in the
Preparation of Purified Guaiacum Resin (1). 2.0 liters of a mixed solution
of methanol/H.sub.2 O (6:4) was added to about 70 g of the resultant dried
matter. 100 ml of the solution was once poured at room temperature into a
column having a diameter of 5 cm and a height of 50 cm and containing
octadecylsilane-treated silica gel (C.sub.18) which had been preliminarily
equilibrated with a mixed solution of 1.2 mM sodium octanesulfonate
aqueous solution/H.sub.2 O (6:4), followed by passing 100 ml/minute of a
mixed solution of 1.2 mM sodium octanesulfonate aqueous solution/H.sub.2 O
(6:4) to separate by elution of impurities. Thereafter, constituents
contained in the column were eluted with methanol. For detection, a UV ray
with 280 nm was used. The fractioneluted with the methanol was evaporated
to dryness in an evaporator to obtain about 0.5 g of purified guaiacum
resin (3).
Preparation of Purified Guaiacum Resin (4)
Natural guaiacum resin was pretreated in the same manner as in the
Preparation of Purified Guaiacum Resin (1). 2.0 liters of a mixed solution
of methanol/H.sub.2 O (6:4) was added to about 70 g of the resultant dried
matter. This solution was set in a high-speed liquid chromatograph for
collection and 100 ml of the solution was once poured at room temperature
into a column having a diameter of 5 cm and a height of 50 cm and
containing butyl-chemically combined silica gel (C.sub.18) which had been
preliminarily equilibrated with a mixed solution of methanol/H.sub.2 O
(6:4), followed by passing 100 ml/minute of a mixed solution of
methanol/H.sub.2 O (6:4) to separate by elution of impurities. Thereafter,
constituents contained in the column were eluted with acetonitrile/H.sub.2
O (6:4). For detection, a UV ray with 280 nm was used. The fraction eluted
with the acetonitrile/H.sub.2 O was evaporated to dryness in an evaporator
to obtain about 0.5 g of purified guaiacum resin (4).
Confirmation of Purified Guaiacum Resin
The first and second constituents and the third and fourth constituents
collected according to the procedure of the Preparation of Purified
Guaiacum Resin (1) were tested in the following manner. The groups
containing the respective constituents were, respectively, developed on a
silica gel-formed plate (F-254) with a mixed solution of
toluene/dioxane/glacial acetic acid (90/25/10), over which an aqueous
solution of peroxidase-H.sub.2 O.sub.2 was sprayed. As a result, it was
confirmed that among a plurality color bands of the crude guaiacum resin,
a group of substances which assumed a reddish purple color and decolored
within a short time after the coloration existed in the second constituent
group. Further, a group of substances which assumed a blue color and were
stable after the coloration belonged to the third constituent group. A
group of substances which were slightly low in color density but were able
to develop a color in different ranges of concentration belonged to the
fourth constituent group. The first constituent group was confirmed to
contain only small amounts of color-developing substances. For the third
and fourth constituent groups, the bands observed under the UV light were
substantially coincident with color bands.
2 ml of a 10% H.sub.2 O.sub.2 aqueous solution, 4 ml of ethanol and 50 mg
of peroxidase of a horseraddish were charged into distilled water to make
9.9 ml of a solution. To the solution was added 0.1 ml of a solution of 2
mg of the purified guaiacum resin (1) in 20 ml of ethanol. 30 seconds
after rapid mixing of the solutions, the mixed solution was subjected to
measurement of a transmittance at 60 nm in a 1 cm cuvette to obtain a
specific absorptivity, E.sup.1%.sub.1 cm, of 300.
REFERENCE 1
Preparation of Purified-by-Normal-Phase Guaiacum Resin
100 g of natural guaiacum resin was dissolved in 150 ml of acetone, to
which 1.5 liters of toluene was added under agitation to filter the
resultant precipitate (about 20 g) by suction. The resultant filtrate was
concentrated in vacuum and dried to obtain about 70 g of a dried matter in
this pretreatment.
500 ml of a mixed solution of n-heptane/acetic ester (2:5) was added to the
dried matter while heating. 100 ml of the solution was added to a silica
gel column (diameter 8 cm, height 70 cm) which had been preliminarily
equilibrated with a mixed solution of n-heptane/acetic ester (2:5),
followed by separation by elution with a mixed solution of
n-heptane/acetic ester (2:5). 100 ml of the solution was used for each
elution. The respective fractions were checked by thin layer
chromatography to collect desired fractions, followed by concentration to
100 ml. The concentrate was mixed with 2 ml of n-hexane for
recrystallization to obtain about 3.5 g of purified guaiacum resin.
Confirmation of Purified-by-normal-phase chromatography Guaiacum Resin:
The thus obtained purified guaiacum resin was developed on a silica
gel-formed plate with a mixed solution of toluene/dioxane/glacial acetic
acid (90:25:10), after which an aqueous solution of peroxidase-H.sub.2
O.sub.2 was sprayed to cause a blue color to be developed, thereby
obtaining an Rf value of 0.45.
Isolation of a First Constituent in Purified Guaiacum Resin
3 g of the purified-by-normal-phase chromatography guaiacum resin was added
to and dissolved in 100 ml of a mixed solution of methanol/H.sub.2 O
(6:4). The solution was set in a high-speed liquid chromatograph for
collection and poured at room temperature into an octadecylsilane-treated
silica gel (C.sub.18) column equilibrated with a mixed solution of
methanol/H.sub.2 O (6:4) and having a diameter of 5 cm and a height of 50
cm. Thereafter, a mixed solution of methanol/H.sub.2 O (6:4) was passed at
a rate of 100 ml/minute to separate impurities by elution, followed by
elution of a constituent retained in the column with methanol. For a
detector, an UV ray of 280 nm was used.
The fraction of a short retention time (0-80 minutes) eluted with the mixed
solution of methanol/H.sub.2 O (6:4) was evaporated to dryness in an
evaporator to obtain about 1.0 g of the first constituent in the
purified-by-normal-phase chromatography guaiacum resin. It was confirmed
that when the purified-by-normal-phase chromatography guaiacum resin was
isolated by the purifying method of the invention, the second constituent
group (retention time: 80-160 minutes) was removed from the purified
guaiacum resin eluted with methanol.
EXAMPLE 2
Fabrication and Evaluation of Performance of Inspection
Articles for Detection of Glucose (1)
The purified-by-reversed-phase chromatography guaiacum resin obtained in
Example 1 and the purified-by-normal-phase chromatography guaiacum resin
and the first constituent group alone thereof obtained in Reference 1 were
mixed with an ink in amounts indicated below, thereby making inspection
articles for detection of glucose. It will be noted that the purified
guaiacum resins of Example 1 have little difference in performance and are
merely referred to as purified-by-reversed-phase chromatography guaiacum
resin.
TABLE 1
______________________________________
Mixing Ratio
______________________________________
Inventive purified-by-reversed-phase
4.8 parts
Product chromatography by weight
guaiacum resin (1)
Control A crude guaiac resin
4.8 parts
by weight
Control B purified-by-normal-phase
4.8 parts
chromatography by weight
guaiacum resin
Control C purified-by-reversed-phase
4.8 parts
chromatography by weight
guaiacum resin and
first constituent group
1.6 parts
by weight
Control D purified-by-reversed-phase
4.8 parts
chromatography by weight
guaiacum resin and
first constituent group
4.8 parts
by weight
______________________________________
The guaiacum resins indicated in Table 1 were used and an ink composition
of the following formulation for detection of glucose was finely dispersed
by means of a homogenizer, followed by printing on a 300 micrometer thick
white polystyrene sheet by screen printing in the form of a quadrilateral
with each side of 5 mm. The screen plate used had a total thickness of a
80 mesh resist and a screen gauze of 130 micrometers.
Ink Composition for Detection of Glucose
______________________________________
Glucose oxidase 3.6 parts
(Grade II available from Toyobo)
by weight
Peroxidase 2.4 parts
(Grade III available from Toyobo)
by weight
Guaiacum resin predetermined amounts
(indicated in Table 1)
Sorbitan monolaurate 7.2 parts
(Span 20 available from Kao Co., Ltd.)
by weight
L-ascorbyl stearate 0.48 parts
by weight
Citric acid 2.8 parts
by weight
Sodium citrate 11.0 parts
by weight
Polyvinylpyrrolidone (Kolidon 90,
12.6 parts
from BASF) by weight
Polyvinylbutyral (Eslek BX-1, from
2.25 parts
Sekisui Chem. Co., Ltd.)
by weight
Fine cellulose powder (Avicel TG-D,
171 parts
from Asahi Chem. Co., Ltd.)
by weight
n-Amyl alcohol 228 parts
by weight
Butylcellosolve acetate
33.5 parts
by weight
______________________________________
The resultant print was dried at 60.degree. C. for 40 minute, after which
it was cut in the form of a flat dipstick to obtain an inspection article
for detection of glucose.
Performance Test (1)
The inspection articles using the inventive product and the controls A, B,
C and D were immersed in specimens of normal urine, and normal urine
dissolving 25 mg/dl, 50 mg/dl, 100 mg/dl, 250 mg/dl and 500 mg/dl of
beta-D-glucose and immediately removed from the specimens. The time
required for coloration and a color tone at the inspection portion were
checked for each article. The results are shown in Table 2.
TABLE 2
__________________________________________________________________________
Concentration
Normal
25 mg/
50 mg/
100 mg/
250 mg/ 500 mg/
of Glucose
urine
dl dl dl dl dl
__________________________________________________________________________
Inventive
-- +/20
++/20
+++ ++++ +++++
Product sec.
sec. /20 sec.
/20 sec. /20 sec.
Control A
-- -- -- +/1 ++/1 +++
min. min. /2 min.
Control B
-- -- +/40 ++.about.+++
++++ +++++
sec. /40 sec.
/40 sec. /40 sec.
Control C
-- -- +/40 ++.about.+++
++++ +++++
sec. /40 sec.
/40 sec. /40 sec.
Control D
-- -- -- ++/1 +++.about.++++
+++++
min. /1 min. /1 min.
__________________________________________________________________________
(Color density +n/time before coloration)
The color tone in the Table was determined by comparison with a color table
under a standard light source. The purified guaiacum resin of the
invention is superior in sensitivity and coloration speed to the crude
guaiacum resin and the guaiacum resins purified by the known method. It
was found that the first constituent group which could be removed by the
purification method of the invention lowered the sensitivity of the
inspection article to a substantial extent.
After the inspection articles had been sealed in a glass container and
stored at 40.degree. C. for 12 months, they were tested in the same manner
as described above. As a result, it was found that the inventive article
and the controls B, C and D were uniform, clear and stable similar to
those before the storage and could detect glucose in specimens in the same
manner as before storage. However, when the control A was tested
similarly, the coloration at the inspection reagent portion became poorer
in uniformity and clearness as compared with that before the storage. It
was difficult to determine a concentration of glucose in specimens. From
this, it was confirmed that the second constituent group was an unstable
constituent.
Performance Test (2)
The inspection sticks for detection of glucose using the inventive product
and the controls A, B, C and D were each immersed in an aqueous solution
dissolving 500 mg/dl of beta-D-glucose and immediately removed, followed
by allowing to stand for 1 minute to check occurrence of color shading to
such an extent of not less than 10% of the inspected portion. 100 sticks
were subjected to measurement of a rate of occurrence of the color shading
with the results shown in Table 3.
TABLE 3
______________________________________
Inventive Control Control Control
Control
Product A B C D
______________________________________
Frequency
2/100 51/100 15/100 9/100 27/100
of Occur-
rence of
Color
Shading
______________________________________
From this, it was found that the removal of the first constituent group and
the collection of the color constituents ensured good uniform
characteristics.
EXAMPLE 3
Fabrication and Evaluation of Performance of Inspection Articles for
Detection of Glucose
Inspection articles of the impregnation-in-filter type containing the
purified-by-reversed-phase chromatography guaiacum resin obtained in
Example 1 were fabricated in the following manner.
A filter paper (No. 526, from Toyo Filter Paper Co., Ltd.) was impregnated
with a solution of the following formulation and dried at 60.degree. C.
______________________________________
Glucose oxidase (Grade II, available
0.72 parts by weight
from Toyobo Ltd.)
Peroxidase (Grade III, from Toyobo Ltd.)
0.48 parts by weight
Purified-by-reversed-phase chromatography
0.96 parts by weight
guaiacum resin
Sorbitan monlaurate (Span 20, from Kao
1.44 parts by weight
Co., Ltd.)
L-ascorbyl stearate 0.10 part by weight
Citric acid 0.56 parts by weight
Sodium citrate 2.20 parts by weight
Polyvinylpyrrolidone (Kodolin
1.26 parts by weight
90, from BASF)
Ethanol 30.0 parts by weight
Water 70.0 parts by weight
______________________________________
The dried filter was cut into 5 mm square pieces and bonded on a 300
micrometer thick white polystyrene sheet by means of a double-coated
adhesive tape, followed by cutting into a stick to obtain an inspection
article for detection of glucose. Normal urine, and normal urine
dissolving beta-D-glucose at concentrations of 50 mg/dl, 100 mg/dl, 250
mg/dl, 500 mg/dl and 2,000 mg/dl were provided as specimens, in which the
sticks were immersed, immediately removed and allowed to stand for 1
minute to observe a color tone of the inspection reagent portion. The
color of the inspection reagent portion was uniform and clear and the
color density increased in a stepwise manner with an increase of the
concentration of glucose in the specimen. Thus, the concentration of
glucose in the specimen could be clearly judged within the above range.
The colored inspection article was allowed to stand for 3 minutes with no
change in color tone being recognized.
The purified-by-reversed-phase chromatography guaiacum resin in the
composition was varied in the following manner, with the result that the
color tone of the inspection portion and the time required for coloration
were as follows.
TABLE 4
__________________________________________________________________________
Glucose Conc. Normal
50 mg/dl
100 mg/dl
250 mg/dl
500
__________________________________________________________________________
mg/dl
Inventive
purified-by-reversed-phase chromatography
-- +/30 sec
++/30 sec
+++/30 sec
++++/30 sec
Product:
guaiacum resin
0.96 parts by weight
Control E:
purified-by-normal-phase chromatography
-- +/40 sec
++/40 sec
+++/40 sec
++++/40 sec
guaiacum resin
0.96 parts by weight
Control F:
crude guaiacum resin -- -- +/40 sec
++/40 sec
+++.about.++++
0.96 parts by weight /40 sec
Control G:
purified-by-reversed-phase chromatography
-- +/40 sec
++/40 sec
+++/40 sec
++++/40 sec
guaiacum resin
0.96 parts by weight and
first constituent group
0.32 parts by weight
Control H:
purified-by-reversed-phase chromatography
-- -- +/40 sec
++.about.+++
++++/40 sec
guaiacum resin /40 sec
0.96 parts by weight and
first constituent group
0.96 parts by weight
__________________________________________________________________________
There was no difference of sensitivity of the inspection articles of the
controls E and G from that of the inventive product. However, it was found
that the sensitivity of the inspection article of the control H which
included a large amount of the first constituent group was lowered. The
inventive product is substantially more excellent than the controls H and
G in the sensitivity of the inspection because the inventive product is
superior in coloration speed to the controls E and G.
EXAMPLE 4
Fabrication of Inspection Articles for Detection of Occult Blood
Among ingredients of the following composition for detection of occult
blood, cumene hydroperoxide and 6-methoxyquinoline used as a sensitizer
were microcapsuled with use of gum arabic, to which other ingredients were
added, thereby obtaining a composition for occult blood. Subsequently, the
composition was uniformly dispersed by means of a homomixer and printed on
a 300 micrometer thick white polystyrene sheet by screen printing to form
an inspection reagent portion in the form of a square with each side of 5
mm. After the printing, it was dried at 60.degree. C. for 40 minutes. The
screen ruling of a plate used for the printing was 80 mesh and the total
thickness of a resist and a screen gauze was 130 micrometers. After
drying, the sheet was cut into stick pieces for inspection.
______________________________________
Composition for Occult Blood:
______________________________________
Cumene hydroperoxide 3.6 parts by weight
6-Methoxyquinoline 1.0 part by weight
(these two encapsulated)
Gum arabic 9.4 parts by weight
Purified guaiacum resin
(Example 1-(1)) 1.5 parts by weight
Citric acid 0.56 parts by weight
Sodium citrate 2.2 parts by weight
Laurylsulfate triethanolamine
1.62 parts by weight
Polyethylene glycol 2.52 parts by weight
Polyvinylbutyral 3.6 parts by weight
(Eslek BX-1, from Sekisui Chem. Co., Ltd.)
Cellulose fine powder 22.4 parts by weight
(Avicel TG-D, from Asahi Chem. Co., Ltd.)
n-Amyl alcohol 39.7 parts by weight
Butylcellosolve acetate
13.0 parts by weight
______________________________________
The following four specimens were provided.
(1) Normal urine
(2) Urine containing 0.06 mg/dl of human hemoglobin (Sigma Co., Ltd.)
(3) Urine containing 0.15 mg/dl of human hemoglobin (Sigma Co., Ltd.)
(4) Urine containing 0.75 mg/dl of human hemoglobin (Sigma Co., Ltd.)
The inspection test pieces were immersed in the above specimens (1) to (4)
and immediately removed, followed by allowing to stand for 1 minute to
check the color of the inspection reagent portion.
The colors of the inspection reagent portions were uniform and clear and
the color density increased in a stepwise manner with an increase of the
human hemoglobin concentration in the specimens. The concentration of the
occult blood in the specimen could be appreciably judged within the above
range. The colored test pieces were allowed to stand for 5 minutes, after
which no change in the color could be recognized.
The test pieces for the inspection were sealed in a glass container and
preserved at 40.degree. C. for 12 months, followed by testing in the same
manner as described above. The color of the inspection reagent portion was
uniform, clear and stable as before and the concentration of human
hemoglobin in specimens could be clearly judged.
EXAMPLE 5
Fabrication of Inspection Articles for Detection of Urea Nitrogen
For detecting of urea nitrogen in serum, inspection articles were made in
the same manner as in Example 2 except that the following ink composition
for detection of urea nitrogen was used instead of the ink composition for
detection of glucose.
______________________________________
Ink Composition for Detection of Urea Nitrogen:
______________________________________
Urease (Grade II, from Toyobo Ltd.)
3.6 parts by weight
Peroxidase (Grade III, from Toyobo Ltd.)
2.4 parts by weight
Purified guaiacum resin (Example 1-(1))
4.8 parts by weight
Sorbitan monolaurate (Span 20, from Kao
7.2 parts by weight
Co., Ltd.)
Citric acid 2.0 parts by weight
Sodium citrate 11.0 parts by weight
Polyvinylpyrrolidone (Kolidon, from
12.6 parts by weight
BASF)
Polyvinylbutyral (Eslek BX-1, from Sekisui
2.25 parts by weight
Chem. Co., Ltd.)
Cellulose fine powder (Avicel, from Asahi
171 parts by weight
Chem. Co., Ltd.)
n-Amyl alcohol 228 parts by weight
Butylcellosolve acetate
33.5 parts by weight
______________________________________
The resultant inspection article was immersed in a serum obtained by
centrifugal separation of the human blood and removed therefrom
immediately after the immersion, and allowed to stand for about 1 minute
to check, whereupon it was found that the reagent layer assumed a blue
color. Upon comparison with a standard color comparison table, it was
found that about 10 mg/dl of urea nitrogen was contained.
EXAMPLE 6
Fabrication of Inspection Article for Detection of Total Cholesterol
For detection of the total cholesterol in a serum, the general procedure of
Example 2 was repeated except that an ink composition for detection of the
total cholesterol was used instead of the ink composition of detection of
glucose, thereby obtaining an inspection article.
______________________________________
Ink Composition for Detection of Total Cholesterol:
______________________________________
Cholesterol esterase (Grade III, from
1.6 parts by weight
Toyobo Ltd.)
Cholesterol oxidase (Grade III, from
1.6 parts by weight
Toyobo Ltd.)
Peroxidase (Grade III, from Toyobo Ltd.)
2.4 parts by weight
Purified guaiacum resin (Example 1-(1))
4.8 parts by weight
Sorbitan monolaurate (Span 20, from Kao
7.2 parts by weight
Co., Ltd.)
Monosodium phosphate 6.0 parts by weight
Disodium phosphate 9.0 parts by weight
Polyvinylpyrrolidone (Kolidon 90,
12.6 parts by weight
from BASF)
Polyvinylbutyral (Eslek BX-1, from
2.25 parts by weight
Sekisui Chem. Co., Ltd.)
Cellulose fine powder (Avice), from Asahi
171 parts by weight
Chem. Co., Ltd.)
n-Amyl alcohol 228 parts by weight
Butylcellosolve acetate
33.5 parts by weight
______________________________________
The obtained inspection article was immersed in a serum obtained by
centrifugal separation of an arbitrary human blood and removed therefrom
immediately after the immersion, and allowed to stand for about 2 minutes.
It was found that the reagent layer assumed a blue color, and the
comparison with a standard color comparison table revealed that the serum
contained a total cholesterol content of about 200 mg/dl.
According to the invention, passage through a column packed with a gel for
reversed phase chromatography permits isolation and elution of impurities
impeding color development and contained in natural guaiacum resin. By
isolation and elution of constituent groups effective for color
development of an inspection article which is used to the detection of
glucose or occult blood in body fluids, a purified guaiacum resin
containing constituents or ingredients capable of color development in a
wide range of concentration can be obtained. In the practice of the
invention, the separability is very good and re-utilization of the column
is possible with good economy. Inspection articles using the purified
guaiacum resin obtained according to the method of the invention for the
purpose of detection of glucose and occult blood in body fluids are
substantially free of any impurities impeding color development. Thus, the
coloration during the course of diagnosis is clear. Moreover, since a
plurality of color-developing constituents are contained, the color
develops in high sensitivity and in a stepwise manner to a high
concentration. The inspection article has high storage stability.
Especially, when an inspection article is obtained by coating a
non-aqueous composition onto a substrate, it ensures uniform color
development and high sensitivity.
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